Compositions and methods for cleaning contaminated solids and liquids

ABSTRACT

The present invention relates to compositions and methods for the remediation of contaminated solids and liquids. In particular, embodiments of the present invention relate to the bioremediation of solids and liquids by a composition comprising a biocatalyst or mixture of biocatalysts. The present invention also relates to methods for producing the bioremediation compositions and methods for applying the bioremediation compositions to contaminated sites, including treatment, storage, and disposal facilities, as well as various contaminated water sources, such as aquifers and reservoirs.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/111,409, filed Aug. 24, 2018 (now U.S. Pat. No. 10,906,075,issuing Feb. 2, 2021), which is a continuation-in-part of U.S. patentapplication Ser. No. 15/196,212, filed Jun. 29, 2016 (now U.S. Pat. No.10,065,224, issuing Sep. 4, 2018), which is a continuation of U.S.patent application Ser. No. 14/456,912, filed Aug. 11, 2014 (now U.S.Pat. No. 9,403,198, issued Aug. 2, 2016), which claims the benefit ofU.S. Provisional Patent Application Ser. No. 61/864,040, filed Aug. 9,2013, the entire content of which is hereby incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for theremediation of contaminated solids and liquids, in particular thebioremediation of solids and liquids by a composition comprising abiocatalyst or mixture of biocatalysts.

BACKGROUND OF THE INVENTION

Chemical contamination of the environment, particularly of soil andgroundwater is currently a widespread problem that is prevalent in manyparts of the industrialized world. Industrial pollution has contaminatedmillions of acres of soil and associated aquifers. Over the past severaldecades worldwide production, processing, storage, transportation andutilization of synthetic and naturally occurring chemical substances hasled to the introduction of significant quantities of hazardous materialsinto the environment. Unintentional spillage of petroleum, industrialsolvents, food and animal wastes and other substances has been caused,for example, by weathering, chemical corrosion and accidental damage topipes, storage vessels, processing equipment, transportation vehicles,etc. Deliberate acts and carelessness have also contributed to therelease of hazardous substances into the environment. The spillage ofsuch materials has resulted in large numbers of polluted sites andenormous volumetric quantities of soil and groundwater which have beencontaminated with hazardous substances. Soil contamination can causeextensive damage to the local ecosystem by accumulating in the tissue ofanimals and plants, and by causing death thereto and/or mutation to theprogeny thereof. Such contamination can also present a serious healththreat to humans and, in extreme cases, can render the contaminated areaunsuitable for human habitation. In many cases, contaminated sites canpose a danger to adjacent property, such as by entrainment of hazardoussubstances by local groundwater flow, and local laws frequently mandateremediation prior to the sale or lease of property wherein the soil hasbeen contaminated with hazardous materials.

Unfortunately, clean up of such contaminated sites and spills isextraordinarily difficult, and can be extremely labor intensive, costlyand time consuming. Oil spills, especially involving water, areparticularly troublesome to treat, as are oil producing well sitescontaminated with crude oil. Existing cleaning technologies were oftendeveloped prior to the current environmentally-conscientious market. Assuch, environmentally harmful potentials such as runoff, toxicity,acid-base balances, and other downstream effects were seen in existingproducts. Technological improvements to existing cleaning productsremained minor since large companies failed to recognize the growingmarket need for a suitable cleaning product applicable to soils, water,and hard surfaces.

The biological treatment or bioremediation of waste water, soil, oilspills, refinery waste, and other contaminants has been attempted in thepast, but most technologies are fraught with complications anddisadvantages. Some of these efforts have attempted to utilize bacteria,fungi or other microbes to biodegrade the contaminants, into moreenvironmentally friendly materials, but most technologies haveaccompanying disadvantages and limitations.

Further, various microbial products were introduced with the concept ofdegrading the contaminants rather than simply transferring the problemfrom one site to another. To this end, enzymes were often added toexisting products. However, since enzymes only perform within narrowlydefined ranges of contaminants, the enzymes often ceased working oncethe contaminants were only partially degraded; leaving behind residueand secondary contaminants that could be as problematic as the originalcontaminant.

For example, European Patent No. 1352694, which is incorporated byreference herein in its entirety, discloses a method for thebioremediation of soils using a compost material derived from plantmaterial, biological sludges, urban waste, animal manure, andcombinations of bacteria, and/or molds in a liquid broth. This solutionhas some potential disadvantages such as difficult raw material handlingand a final remediation agent that is limited to the treatment of soils.In addition the composting step may require a significant amount of timeto produce the target remediation product, and the wide variation in thecomposting raw materials will likely result in broad variability in thebioremediation agent's final bioactivity and the composition of itsfinal microbial flora.

U.S. Pat. No. 5,265,674 (“the '674 Patent”) describes a method for theremediation of aquifers comprising injecting a liquid oil into thecontaminated site, wherein the oil may further comprise microorganismsand nutrients. This method may be problematic if the oil itself does notdegrade and subsequently accumulates in the aquifer. Further the methodis somewhat limited in that it appears to be limited to the treatment ofcontaminated water sources. Finally, this method is applied as a liquid,which has spill hazards and complex, costly application methods. PCTApplication WO 99/46210 and U.S. Patent Application Publication No.2013/0023037 also describe composting methods, and both potentiallysuffer from the same disadvantages as the ′674 Patent.

Some methods for remediating soils that utilize in situ techniques suchas aeration, venting and air sparging are generally limited tocontaminants having a relatively high vapor pressure. Compounds such aspolycyclic aromatic hydrocarbons, which have a low-vapor pressure,cannot be successfully removed by volatilization. Moreover, conventionalbioremediation techniques utilizing indigenous microorganisms alone orin combination with genetically altered exogenous microorganisms is notalways effective for degrading certain types of recalcitrantcontaminants which are strongly resistant to biodegradation. Geneticallyengineering microbes is also time consuming and expensive and may resultin a strain that is capable of achieving a desired metabolic function,but is weakened in other key metabolic areas, resulting in a strain thatis ineffective or cost-prohibitive in large-scale production and use.

In addition, many of the present techniques for remediating soils andwater take long periods of time to reduce the contaminants to acceptableor non-detectable limits. Faster treatment times of larger contaminatedareas or volumes is a long-standing need in industry, that has thepotential to reduce the cost of bioremediation, as well as the lostopportunity costs of land and resources that cannot be used due theircontaminated state.

Thus, while various known techniques are available for the disposal orreclamation of contaminated soil, such methods do not generally providea practical, affordable technology for remediating soil and watersources in reasonable periods of time. Accordingly, there is a need forsimple, inexpensive, environmentally acceptable methods and means forremediating soils and water, within time spans on the order of days,instead of many months or even years.

SUMMARY OF THE INVENTION

It is therefore one aspect of embodiments of the present invention toprovide a remediation biocatalyst composition that is effective atremoving a wide variety of contaminants from contaminated solids andliquids, in particular contaminated soils and water supplies, such asground water and aquifers. It is an aspect of embodiments of theinvention to provide biocatalyst compositions and methods of use for theremediation of large scale contaminated soil and ground water sites thatquickly reduce the contaminants to acceptable levels, allowing theresources to once again enter normal use or consumption, whereinremediation times are on the order of days to months, rather than on theorder of years. It is another aspect of embodiments of the invention toprovide compositions and methods for the remediation of contaminatedsoils and water supplies that are simple and cost-effective from bothmanufacturing and use perspectives. Another aspect of embodiments of thepresent invention is to provide a partially dry composition, which hasadvantages over liquid compositions including reduced spill hazards andsimpler, less costly application methods. It is yet another aspect ofembodiments of the present invention to provide compositions thatutilize readily available materials, including unwanted industrialby-product streams and/or agricultural by-product streams.

It is one aspect of embodiments of the present invention to provide anew product that not only remediated surfaces esthetically but actuallyreduced or eliminated the organic contaminants responsible for theproblem, and could also be used over a wide variety of applicationsincluding water and soil remediation. Living bacterial systems arecapable of switching on and off enzyme genes to produce a wide varietyof the correct enzymes as the contaminants and the conditions changed.However, no one organism can breakdown all contaminants to the finaldesirable end points (such as water and harmless nitrogen gas). Simplycombining all of the various microbes that can individually breakdownthe various components proved ineffective probably because ofcompetitive interactions between the various species of bacteria.Finding the right microbial “team” is only a portion of the problem withremediation products; there are other requirements that must be met foran environmentally friendly, yet effective, decontamination. Microbesfunction by producing exterior enzymes that breakdown molecules outsideof their cellular walls, which can be transferred into the organism tobe further broken down inside the cells.

Effective storage and shipping of mass-blended microorganisms was alsodifficult and frequently lead to nonviable organisms. Though liquidformulations had some ease of application benefits, their freeze/thawproblems and short cast of available players (amenable to the necessarypreservation techniques) limited their widespread application. Driedbacterial products offered many advantages to problems encountered inthe oncoming “microbial revolution.” If the species can be preserved asa spore, it can withstand harsh shipping and storage conditions and hasan extended life span that can cover centuries. Thus, it is one aspectof embodiments of the invention to provide a dry bioremediation formulathat can be shipped and stored and contain viable organisms.Furthermore, many organisms can be freeze dried which further increasesthe possible array of microbial candidates.

It is one aspect of embodiments of the present invention to provide abioremediation formula with microbes and surfactants. A microbe workingexclusively on the surface of a spill will be too slow in theirbreakdown efforts be effective. Such an application requires thecontaminants to be removed and blended to be more available to themicrobes. With the addition of a surfactant, smaller sphericalsuspensions of contaminants are produced that can be attacked by theappropriate microbes from all directions at once rather than merely on aflat surface.

It is another aspect of embodiments of the present invention to providea bioremediation formula with a co-absorbent to capture and hold theintermediate breakdown byproducts for further degradation. Theseshort-lived secondary intermediates can otherwise escape and avoidfurther degradation. A co-absorbent allows the organisms in the productto continue the break-down pathway to a desirable end-point.

One aspect of embodiments of the present invention is to provide abioremediation formula with an abrasive ingredient to help physicallyremove the contaminant as well as buffers, and other items, known to addcomplimentary properties to the entire product. Microbial candidates,co-absorbents, abrasives, and supporting nutrient are selected for thebioremediation formula with full consideration of environmental impactimplications. Where possible, naturally occurring items (such as cotton,sand, corn and chalk) are selected to meet those needs. Using recycledwaste products, such as fly ash and kiln dust, are also used asenvironmentally friendly components. PCT Patent Publication No. WO03/080787 to Sen et al. discloses using fly ash as an abrasiveingredient in a scouring powder composition and is incorporated byreference herein in its entirety. U.K. Patent Publication No. GB 2 351502 to Salem also disclosing cleaning materials including fly ash and isincorporated by reference herein in its entirety.

The bioremediation composition may be an emulsion comprising multipleingredients. The emulsion can be similar to the one described in U.S.Pat. No. 6,511,954 to Wilburn et al., which is incorporated by referenceherein in its entirety.

Because the action of embodiments of the bioremediation formula resultsin a complex and dynamic living system that is constantly changing, theexact identity of the enzymes and the exact role of any one ingredientat any one time is also changing and may be indeterminable; but theblend of living organisms create a choreography of synergistic livingorganisms all playing together as an effective team to degrade thecontaminant. One embodiment of the formulation enhances thebioavailability to the included microbes.

It is one aspect of embodiments of the present invention to provide abioremediation composition that can work with or without water and thatcan work on water and ice. Thus, in one embodiment, the bioremediationcomposition functions by pulling water out of the atmosphere; therefore,the bioremediation process is accelerated when water is added to thebioremediation composition or when the bioremediation composition isused on water or ice.

It is a further aspect of embodiments of the present invention toprovide a method of producing a bioremediation composition usingbiological hosts to synthesize/accumulate bioproducts such as bacteria,eukaryotic microorganisms (e.g., Saccharomyces cerevisiae), plants,animal cells (e.g., transformed insect cells growing in culture), andanimals. For example, methods by which effector-sensitive RCANAs can beused to facilitate industrial biosynthesis and bioremediation areincluded herein. U.S. Patent Publication No. 2004/0126882 to Ellingtonet al. is incorporated by reference herein in its entirety.

An aspect of the present invention includes a composition for theremediation of contaminated soils or water supplies comprising an oxidephase, a silicate phase, a pH adjustment agent, a densification agent, anutrient, a surfactant, and a biocatalyst, wherein contacting thecontaminated soil or water with said composition results in a visualcolor change of said contaminated soil or water after seven days of saidcomposition being spread on said contaminated soil or water.

A further aspect of the invention includes a composition for theremediation of contaminated liquids or solids comprising an oxide phasebetween about 20 wt % and about 40 wt %, a silicate phase between about5 wt % and about 25 wt %, a pH adjustment agent between about 1 wt % andabout 20 wt %, and a densification agent between about 1 wt % and about20 wt %. The composition further comprises a nutrient between about 1 wt% and about 10 wt %, a surfactant between about 1 wt % and about 10 wt %and a microbial agent between about 10 wt % and about 30 wt %.

Yet another aspect of the present invention includes a composition forthe remediation of contaminated liquids or solids comprising betweenabout 20 wt % and about 40 wt % fly ash, between about 10 wt % and about20 wt % Portland cement kiln dust, between about 5 wt % and about 15 wt% sand, between about 0.5 wt % and about 4 wt % milled whole kernelcorn, between about 0.5 wt % and about 4 wt % milled cotton hulls,between about 5 wt % and about 15 wt % calcium carbonate, between about2 wt % and about 10 wt % grout, between about 1 wt % and about 10 wt %detergent, and between about 10 wt % and about 30 wt % of biocatalyst.The biocatalyst comprises between about 5 wt % and about 15 wt %Bacillus amyloliquifaciens, between about 5 wt % and about 15 wt %Bacillus atophaeus, between about 5 wt % and about 15 wt % Bacillusbenzeovorans, between about 5 wt % and about 15 wt % Bacillus cereus,between about 5 wt % and about 15 wt % Bacillus lichenformis, betweenabout 5 wt % and about 15 wt % Bacillus megarterium, between about 20 wt% and about 25 wt % Bacillus subtilus, between about 5 wt % and about 15wt % Bacillus polymyxa, between about 5 wt % and about 15 wt %Micrococcus flavus, and between about 5 wt % and about 15 wt %Micrococcus conglomerates.

In some embodiments of the present invention, the composition mayfurther comprise a particle size of less than 0.125 inches. In someembodiments of the present invention, the composition may furthercomprise a moisture content of less than 15 wt %. In one embodiment ofthe invention, contacting the contaminated solids or liquids with saidcomposition results in a visual color change of said contaminated solidsor liquids after seven days of said composition being spread on saidcontaminated solids or liquids

In various embodiments of the present invention a bioremediationcomposition is provided with traces of perfumes, charcoal, and/or odorabsorbing compounds.

In one embodiment, the composition comprises a urea hydrochloride, asurfactant, and a glycol ether. Glycol ethers are miscible with waterand retain their solvent properties in water solutions. The compositionmay also include a corrosion inhibitor to protect the surface beingcleaned. Further, essential oils (such as terpene), terpenehydrocarbons, petroleum distillates, and/or di-basic esters may be addedto the composition.

In some embodiments, the composition is in the form of a solid, gel,solution, paste, or a powder.

This Summary of the Invention is neither intended nor should it beconstrued as being representative of the full extent and scope of thisdisclosure. Moreover, references made herein to “the present invention”or aspects thereof, should be understood to mean certain embodiments andshould not necessarily be construed as limiting all embodiments to aparticular description. The present invention is set forth in variouslevels of detail in the Summary of the Invention as well as in theDetailed Description and Examples and no limitation as to the scope isintended by either the inclusion or non-inclusion of elements,components, etc. in this Summary of the Invention. Additional aspectswill become more readily apparent from the Detailed Description,particularly when taken together with the examples.

DETAILED DESCRIPTION

The following detailed description illustrates the invention by way ofexample and not by way of limitation. This description will clearlyenable one skilled in the art to make and use the invention. To reducethe need to provide extensive disclosure in this application, but toprovide adequate written description of the various devices and methodsencompassed by the numerous embodiments of the present invention,various patents are incorporated herein in their entireties byreference.

References in the specification to “one embodiment”, “an embodiment”,“an example embodiment”, “some embodiments”, etc., indicate that theembodiment described may include a particular feature, structure, orcharacteristic, but every embodiment may not necessarily include theparticular feature, structure, or characteristic. Moreover, such phrasesare not necessarily referring to the same embodiment. Further, when aparticular feature, structure, or characteristic is described inconnection with an embodiment, it is submitted that it is within theknowledge of one skilled in the art to affect such feature, structure,or characteristic in connection with other embodiments whether or notexplicitly described.

As used herein, the term “remediation” refers to a process wherebycontaminants are degraded from a first concentration to a second lowerconcentration. This is typically from a first unacceptable contaminatedconcentration, to a second acceptable concentration that more closelyapproaches a normal, naturally occurring background level. In someembodiments of the present invention, remediation may use livingorganisms or microbes, for example microorganisms, to degrade ortransform environmental contaminants into less toxic forms. This isreferred to as “bioremediation.” Bioremediation is a subset ofremediation.

As used herein, the term “contaminant” refers to any undesirablechemical or compound that is present at some level in a bulk solid,liquid, or gas. Examples of contaminants processed by the presentinvention include, but are not limited to, chemicals, petroleum, oils,toxins, and poisons. Contaminants are typically man-made, manufacturedcompounds including, but not limited to, petroleum products,hydrocarbons, oils, greases, synthetic chemicals, pesticides, herbicidesand the like. Other examples of contaminants that may be treated by someembodiments of the present invention include, but are not limited to,chlorinated solvents, polychlorinated biphenyls, chlorinated phenols,benzene, toluene, ethylebenzene, xylene, and polyaromatic hydrocarbons(PAHs). Still further examples of contaminants include, but are notlimited to, food waste, meat processing waste, animal-by-products,agricultural waste, biological waste, hospital waste, blood productwaste, sewage, algae, and various post-consumer wastes.

As used herein, the term “hydrocarbon” refers to compounds comprisinghydrogen and carbon. The term also refers to compounds comprisinghydrogen, carbon and oxygen, as well as compounds comprising hydrogen,carbon, oxygen and at least one other element.

As used herein, the term “oil” refers to a liquid mixture of a varietyof hydrocarbons. Such mixtures may comprise, for example, volatilecompounds, saturated hydrocarbons, and/or aromatic hydrocarbons.Volatile compounds may comprise low molecular weight compounds, likemethane (natural gas) or propane that are normally gaseous or evaporatevery quickly at room temperature. Saturated hydrocarbons may comprisecompounds with carbon and hydrogen atoms connected only by single bonds.Saturated hydrocarbons can be arranged in straight or branched chains ofup to and even greater than 25 carbon atoms. Saturated hydrocarbons maybe readily remediated although degradability tends to decrease withchain length. Aromatic compounds may comprise compounds that containrings of carbon atoms held together with double bonds between the carbonatoms. The smallest aromatic compounds in petroleum have six carbons insuch a ring structure (e.g., benzene and toluene), but other compoundscontain multiple rings. These are polycyclic aromatic hydrocarbons(PAH). Most aromatic molecules in petroleum have multiple attachedhydrocarbon chains. The smallest aromatic molecules (one- and two-rings)are both volatile and readily biodegraded, even with attachedside-chains. However, four-ring and larger aromatic compounds tend to bemore resistant to remediation. The percentage of PAHs in crude oilvaries, but the ‘priority pollutants’ are present at low levels in crudeoils; they are much more common as a byproduct of burning carbonaceousmaterials such as fuel, coal, wood, tobacco and other materials.Asphaltenes are examples of high molecular weight PAHs that haveadditional chemical side chains attached to their aromatic rings.Asphaltenes are not soluble in water and most organic solvents. The term“oil” also refers to larger molecular weight molecules including, butnot limited to, crude, diesel fuel, jet fuel and other even highermolecular weight compounds. These may be saturated, unsaturated, cyclic,straight-chained or branched molecules.

As used herein, the term “surfactant” refers to nonionic surfactants,cationic surfactants, anionic, and amphoteric surfactants. Examples ofnonionic surfactants that may be utilized in some embodiments of thepresent invention include, but are not limited to, are alkoxylated alkylphenols, amides, amines, ethoxylated or propoxylated higher aliphaticalcohols, and sulphonamides. These surfactants include sorbitan estersof C10 to C22 fatty acids, polyoxyethylene sorbitan esters of C10 to C22fatty acids, polyoxyethylene sorbitol esters of C10 to C22 fatty acids,polyoxyethylene derivatives of C6 to C20 fatty phenols, andpolyoxyethylene condensates of C10 to C22 fatty acids or fatty alcohols.Other suitable nonionic surfactants include sorbitol monolauratepropylene oxide condensates, sorbitol monomyristate propylene oxidecondensates, sorbitol monostearate propylene oxide condensates, dodecylphenol propylene oxide condensates, myristyl phenol propylene oxidecondensates, octylphenyl propylene oxide condensates, nonlyphenylpropylene oxide condensates, stearyl phenol propylene oxide condensates,lauryl alcohol propylene oxide condensates stearyl alcohol propyleneoxide condensates, secondary alcohol propylene oxide condensates such asC14-C15 secondary alcohols condensed with propylene oxide, sorbitantristearate condensed with propylene oxide, sorbitan trioleate condensedwith propylene oxide, and sorbitan trioleate, and polyoxyethylene andpolyoxypropylene analogs of the above surfactants.

Examples of cationic surfactants include, but are not limited to,quaternary ammonium surfactants such as C10 to C22 fatty ammoniumcompounds, C10 to C22 fatty morpholine oxides, propylene oxidecondensates of C10 to C22 fatty acid monoesters of glycerins, the mono-or diethanol amides of C10 to C22 fatty acids, and alkoxylated siloxanesurfactants containing propylene oxide units and/or propylene oxideunits. As is known in the surfactant art, the counterion for quaternaryammonium surfactants is usually a halide, sulfate, or methylsulfate, thechlorides being the most common industrially available compounds. Othersuitable cationic surfactants suitable for use in the present inventioninclude straight chain alkyl fatty amines, quaternary ammonium salts,alkyl-substituted quaternary ammonium salts, alkylaryl-substitutedquaternary ammonium salts, quaternary imidazolinium salts, amine oxides,fatty amine oxides, tri-fatty amine oxides, tri-quaternary phosphateesters, amphoglycinate phosphates, amine acetates, long chain amines andtheir salts, diamines and their salts, polyamines and their salts,polyoxyethylenated long chain amines, and quaternized polyoxyethylenatedlong chain amines.

Examples of anionic surfactants include, but are not limited to, alkalimetal, ammonium and magnesium salts of alpha olefin sulfonates, alkylsulfonates, alkylaryl sulfonates, alkylaryl ether sulfates, alkylethersulfates, sulfated alcohol ethoxylates, taurates, petroleum sulfonates,alkylnapthalene sulfonates, alkylsarcosinates and thealkylsulfosuccinates. Further examples of anionic surfactants include,but are not limited to, sodium lauryl sulfonate, ammonium laurylsulfonate, dodecyl benzene sulfonate, sodium lauryl ether sulfate,diethanolamine lauryl sulfate, ammonium salts of sulfated alcoholethoxylates, sodium cocoyl isethionate, sodium N-methyl-N-oleoyltaurate, sodium N-methyl-N-cocyl taurate, triethanolamine laurylsulfate, disodium monooleamide PEG-2 sulfosuccinate, petroleum sulfonatesodium salt, alkyl napthalene sodium sulfonates, sodium lauroylsarcosinate, and sodium alkyl sulfosuccinate. Other useful anionicsurfactants include sodium or potassium dodecyl sulfate, sodiumtrioleate, sodium or potassium stearyl sulfate, sodium or potassiumdodecyl benzene sulfonate, sodium or potassium stearyl sulfonate,triethanol amine salt of dodecyl sulfate, sodium laurate, sodium orpotassium myristate, and sodium or potassium stearate.

Examples of amphoteric surfactants include, but are not limited to,betaines, sultaines, imidazoline derivatives and the like. Specificamphoteric surfactants useful in the present invention includericinoleamidopropyl betaine, cocamidopropylbetaine, stearyl betaine,stearyl amphocarboxy glycinate, sodium lauraminopropionate,cocoamidopropyl hydroxysultaine, disodium lauryliminodipropionate,tallowiminodipropionate, cocoamphocarboxy glycinate, cocoimidazolinecarboxylate, lauric imidazoline monocarboxylate, lauric imidazolinedicarboxylate, lauric myristic betain, cocoamidosulfobetaine,alkylamidophosphobetain and the like. Further examples of usefulamphoteric surfactants include decyl amino betaine, coco amidosulfobetaine, oleyl amido betaine, coco imidazoline, cocosulfoimidazoline, cetyl imidazoline,1-hydroxyethyl-2-heptadecenylimidazoline, 1-hydroxyethyl-2 mixedheptadecenyl heptadecadienyl imidazoline, and n-coco morpholine oxide.

As used herein, the term “detergent” refers to a surfactant or a mixtureof surfactants. A detergent may assist with the physical interactionbetween two or more unlike components, for example oil and water, bystabilizing the interface between the two components.

As used herein, the term “bleaching agent” refers to a compound whichremoves color, whitens and/or disinfects. As used herein, bleaching mayoccur via oxidation or reduction reactions. Examples of bleaching agentsthat may be utilized in that may be used in some embodiments of thepresent invention include, but are not limited to, sodium hypochlorite,calcium hypochlorite, peroxides, hydrogen peroxide, sodium percarbonate,sodium perborate, sodium dithionite, sodium borohydride, peracetic acid,and benzoyl peroxide. Examples of solid bleaching agents are disclosedin U.S. Pat. No. 6,773,625 to Falk et al. and European PatentApplication Publication No. 0234626, both of which are incorporated byreference in their entirety herein.

As used herein, the term “pH adjustment agent” refers a chemical,compound or material that is used to manipulate or adjust the pH of amixture. Examples of pH adjustment agents that may be used in someembodiments of the present invention include, but are not limited to,acids which may be used to lower the pH of a mixture or composition witha starting value that is relatively high. Alternatively, furtherexamples of pH adjustment agents include, but are not limited to, baseswhich may be used to raise the pH of a mixture or composition with astarting value that is relatively low. pH adjustment may be critical insystems defined primarily by chemical reactions in the absence ofenzymes and microbes. However, pH adjustment may also be important indefining the optimum pH for maximum enzyme activities and microbialgrowth and metabolic rates.

Examples of pH adjustment agents that may be used in some embodiments ofthe present invention include various acids, which may be weak acids,strong acids, mineral acids, or organic acids. A strong acid is definedas an acid that completely disassociates in water, whereas a weak aciddoes not. Examples of acids include, but are not limited to, selenicacid, selenious acid, silicofluoric acid, telluric acid, tellurous acid,tungstic acid, xenic acid, citric acid, formic acid, pyroantimonic acid,permanganic acid, antimonious acid, antimonic acid, hypofluorous acid,phthalic acid, antimonous acid, silicic acid, titanic acid, arsenicacid, perpechnetic acid, hypophosphoric acid, pyrophosphoric acid,hydroarsenic acid, dichromic acid, tetraboric acid, metastannic acid,hypooxalous acid, glutamic acid, cyanic acid, silicous acid, fluorousacid, ferricyanic acid, malonic acid, fluoric acid, hydrocyanic acid,and thiocyanic acid. Additionally, the microbes in bioremediationcomposition may be acid-resistant such that acidic pH adjusting agentscan be used in the composition.

Further examples of pH adjustment agents that may be used in someembodiments of the present invention include various bases, which may beweak bases or strong bases. A strong base is defined as an acid thatcompletely disassociates in water, whereas a weak base does not.Examples of strong bases include, but are not limited to, the hydroxidesof Group I and Group II metals, for example, LiOH, NaOH, KOH and RbOH.Examples of weak bases include, but are not limited to, alanine, ammoniaand methylamine. Still further examples include various carbonates, suchas calcium carbonate and sodium bicarbonate.

As used herein the terms “buffer” and “buffer solution” refer to aqueoussolutions comprising a mixture of a weak acid and its conjugate base ora weak base and its conjugate acid. The pH of a buffer changes verylittle when a small amount of strong acid or base is added to it andthus it is used to prevent changes in the pH of a solution. This isimportant in a number of applications, for example, in chemical systemsor mixtures comprising enzymes and/or microbes wherein the viability,productivity, and/or activity of the enzymes and/or microbes areextremely pH dependent.

As used herein, the term “enzyme” refers to any of numerous proteinsthat cause chemical reactions. In some embodiments of the presentinvention enzymes used may include, but are not limited to,oxidoreductases, transferases, hydrolases, lyases, isomerases andligases. An oxidoreductase catalyzes oxidation/reduction reactionsand/or transfers hydrogen and/or oxygen atoms or electrons from onesubstance to another. Examples of oxidoreductases include dehydrogenaseand oxidase. A transferase transfers a functional group from onesubstance to another, for example methyl, acyl, amino, or phosphategroups. Examples of transferases include transaminase and kinase. Ahydrolase results in the formation of two products from a substrate byhydrolysis. Examples of hydrolases include lipase, amylase, andpeptidase. A lyase results in the non-hydrollytic addition or removal ofgroups from substrates. A lyase reaction may result in the cleavage ofC—C, C—N, C—O, and/or C—S bonds. An example of a lyase includesdecarboxylase. An isomerase results in intramolecular rearrangementswithin a single molecule. Examples of isomerases include isomerase andmutase. A ligase joins two molecules by synthesis of new C—O, C—S, C—Nand/or C—C bonds with the simultaneous breakdown of ATP. An example of aligase includes synthetase.

Further examples of enzymes that may be used in some embodiments of thepresent invention include, but are not limited to, catalases, glucoseoxidases, laccases, fructosyltransferases, glucosyltransferases,amylases, cellulases, lipases, mannanases, pectinases, phytases,proteases, pullulanases, xylanases, pectate lysases, alpha-acetolactatedecarboxylases, and glucose isomerases.

As used herein, the term “microbe” refers to bacteria, yeast, fungi,algae, protozoa, and combinations thereof. In some embodiments of thepresent invention, naturally occurring microbes may be used.Alternatively, genetically engineered microbes may be used. Bacteriathat may be used in some embodiments of the present invention may beaerobic such as Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, andMycobacterium. In some other embodiments the bacteria utilized may beanaerobic. In yet another example, the bacteria utilized may bemethylotrophs, or bacteria that utilize methane for carbon and energy.An example of a bacterium that may be utilized in the present inventionincludes, but is not limited to, Pseudomonas putida which is agram-negative soil bacterium that can digest toulene, a component ofpaint thinner. It is also capable of degrading naphthalene, a product ofpetroleum refining, in contaminated soils as described in U.S. Pat. No.5,536,407 to Petersen, which is incorporated by reference herein in itsentirety herein. Another example is Dechloromonas aromatic, a soilbacteria genus which is capable of degrading perchlorate and aromaticcompounds. Further examples of bacteria include Nitrosomonas europaea,Nitrobacter hamburgensis, and Paracoccus denitrificans which are capableof digesting contaminants found in industrial waste water. Most wastewater treatment systems rely on microbial activity to remove unwantedmineral nitrogen compounds (e.g., ammonia, nitrite, nitrate). Theremoval of nitrogen is a two stage process that involves nitrificationand denitrification. During nitrification, ammonium is oxidized tonitrite by organisms like Nitrosomonas europaea. Then, nitrite isfurther oxidized by microbes like Nitrobacter hamburgensis. In anaerobicconditions, nitrate produced during ammonium oxidation is used as aterminal electron acceptor by microbes like Paracoccus denitrificans.The result is dinitrogen gas. Through this process, ammonium andnitrate, two pollutants responsible for eutrophication in naturalwaters, are remediated.

A further example of a bacterium that may be employed in someembodiments of the present invention is Deinococcus radiodurans which isa radiation-resistant extremophile bacterium that is geneticallyengineered for the bioremediation of solvents and heavy metals. Afurther example of a bacterium that may be utilized in some embodimentsof the present invention is Methylibium petroleiphilum which is capableof digesting methyl tert-butyl ether (MTBE).

A further example of a bacterium utilized in the present inventionNitrosomonas europea. Trihalomethane(s) (THM) contaminated water may beremediated through metabolism of Nitrosomonas europea. Yet anotherexample of a bacterium that may be utilized in some embodiments of thepresent invention is Methylibium petroleiphilum, which is capable ofcompletely mineralizing MTBE. Methylibium petroleiphilum is capable ofconsuming a diverse range of gasoline derivatives as its sole carbonsource, including: methanol, ethanol, toluene, benzene, ethylbenzene,and dihydroxybenzenes.

Still further embodiments of the present invention may utilize bacteriafrom the genus Bacillus. For example, species from the Bacillus genusthat may be used include, but are not limited to, B. alcalophilus, B.alvei, B. aminovorans, B. amyloliquefaciens, B. aneurinolyticus, B.anthracis, B. aquaemaris, B. atrophaeus, B. benzeovorans, B.boroniphilus, B. brevis, B. caldolyticus, B. centrosporus, B. cereus, B.circulans, B. coagulans, B. firmus, B. flavothermus, B. fusiformis, B.globigii, B. infernus, B. larvae, B. laterosporus, B. lentus, B.licheniformis, B. megaterium, B. mesentericus, B. mucilaginosus, B.mycoides, B. natto, B. pantothenticus, B. polymyxa, B. pseudoanthracis,B. pumilus, B. schlegelii, B. sphaericus, B. sporothermodurans, B.stearothermophilus, B. subtilis, B. thermoglucosidasius, B.thuringiensis, B. vulgatis, and B. weihenstephanensis.

Still further embodiments of the present invention may utilize bacteriafrom the genus Micrococcus. For example, species from the Micrococcusgenus that may be used include, but are not limited to, M. antarcticus,M. agilis, N, cohnii, M. endophyticus, M. flavus, M halobius, M.kristinae, M. lactis, M. luteus, M. lylae, M. nishinomiyaensis, M.mucilaginosis, M. roseus, M. mortus, M. sedentarius, M. terreus, M.varians, and M. yunnanensis.

U.S. Pat. No. 5,990,067 to Franssen et al. and U.S. Pat. No. 7,459,421to Bullis et al., both of which are incorporated by reference herein intheir entireties, disclose dry compositions for treating contaminatedconcrete surfaces, wherein the compositions optionally includemicroorganisms including bacteria. Also, incorporated by reference intheir entireties, for written description and enablement purposes, arethe following patents and applications on points related to the use ofbacteria for bioremediation: U.S. Pat. Nos. 5,508,194; 6,503,746;8,444,962; 5,939,086; 5,464,766; 5,536,407; U.S. Patent ApplicationPublication Nos. 2010/0274069; 2003/0100098; 2012/0094360; PCTApplication Nos. WO 94/29242; WO 95/08513; WO 99/66080; WO 2005/042724;WO 2006/018306.

In some embodiments of the present invention, the bioremediationcomposition includes a form of silicate. The silicates are divided intodifferent classes by their structures, such as, hydrous aluminumsilicates or phyllosilicates, which can be used in the bioremediationcompositions. Zeolite, clinoptilolite, and other members of the Zeolitefamily including any within the framework of silicates having anexchanging cation, for example, amicite, shabazite, pistilbite,ferrierite, gobbinisite and mazzite, are absorptive or absorbing agents.Zeolite's color combined with the other ingredients gives it colorationwithout the use of artificial pigments. Zeolite has a chemical name ofpotassium-calcium-sodium-aluminosilicate, One producer of this is BearRiver Zeolite Corporation, in Thompson Falls, Mont. It is part of thehydrous aluminum silicates chiefly found in igneous rocks andcharacterized by a ready loss or gain of water. Zeolites are used asmolecular sieves to separate mixtures because they are capable ofselective absorption and are a microporous material that is capable ofabsorbing and encapsulating other agents. One of Zeolites' functions issimilar to that of microorganisms, but instead of digestingcontaminants, it imprisons them, They have a high ion exchange capacityand can be used to separate petrol, benzene, and toluene from low graderaw materials, such as, coal and methanol.

Additionally, the following patents are incorporated by reference forfurther enablement and disclosure: U.S. Pat. No. 7,071,153 to Lewis etal.; U.S. Pat. No. 6,969,699 to Sen et al.; U.S. Pat. No. 7,658,805 toNetherton; U.S. Pat. No. 8,206,062 to Hoag et al.; U.S. Pat. No.6,511,954 to Wilbur et al.; U.S. Pat. No. 8,197,605 to Laffitte et al.;and U.S. Pat. No. 8,133,855 to Dreilinger et al.

Fungi are well-suited for PAH degradation and higher molecular weightsubstances. They also function well in non-aqueous environments wherehydrophobic PAHs accumulate; a majority of other microbial degradationoccurs in aqueous phase. They can function in the very low-oxygenconditions.

A specific example of a fungus that may be utilized in some embodimentsof the present invention is the lignin-degrading white rot fungus,Phanerochaete chrysosporium, which exhibits a strong potential for thebioremediation of pesticides, polyaromatic hydrocarbons, PCBs, dioxins,dyes, TNT and other nitro explosives, cyanides, azide, carbontetrachloride, and pentachlorophenol. White rot fungi degrade ligninwith nonselective extracellular peroxidases, which can also facilitatethe degradation of other compounds containing similar structure tolignin within the proximity of the enzymes released.

Other examples of fungi that may be used in some embodiments of thepresent invention include Armillaria, Heterobasidion annosum, Serpulalacrymans, Lenzites trabea, Fibroporia vaillentii, and Sporotrichumpulverulentum.

Also, incorporated by reference in their entireties on points related tothe use of fungi for bioremediation for written description andenablement purposes are the following: U.S. Pat. Nos. 5,476,788;5,486,474; PCT Application Publication No. WO 2006/136177. Also,incorporated by reference in their entireties on points related to theuse of fungi and bacteria for bioremediation for written description andenablement purposes are U.S. Pat. No. 6,194,197 and PCT ApplicationPublication No. WO 00/41976.

In some embodiments of the present invention, yeast may be used in abioremediation composition and/or method to remove contaminants from asolid or a liquid. Yeasts are eukaryotic microorganisms classified inthe kingdom Fungi, with 1,500 species currently described (estimated tobe 1% of all fungal species). One example of a yeast that may be used insome embodiments of the present invention is Yarrowia lipolytica whichis capable of degrading palm oil and mill effluent, TNT, and otherhydrocarbons such as alkanes, fatty acids, fats and oils.

As used herein, the term “nutrient” refers to any chemical compound ormatter that provides the material needed to enable microbe metabolism,growth, viability and productivity. As with any other living organism,microbes such as bacteria, fungi and yeast require nutrients to survive.For example, microbes require a carbon source, which can be obtainedfrom, sugars, alcohols and other organics. However, microbes alsorequire a source of nitrogen, amino acids, various salts and essentialelements in order to grow and metabolize normally. Further examples ofnutrients may include, but are not limited to, agar, starch, sugars,molasses, yeast extract, corn-steep liquor, spent fermentation mash,dried distiller grain solids (DDGS), milled corn, corn flour, cottonhulls, wheat straw, corn stover, rice, grains, leaves, dung, manure,residential yard waste, bagasse, bark, saw dust, and wood chips.

As used herein, the term “densification agent” refers to any solid orliquid additive that raises the bulk density of a first mixture, whenthe densification agent is added to and mixed with the first mixture. Asused herein, the term “binding agent” refers to any solid or liquidmaterial that assists with binding together two or more components of amixture. A binding agent, among other things, may be utilized to assistwith transporting a mixture from one point to another, assist with theapplication of a composition to a target area, surface, solid, liquid,etc., and assist with dust control and/or mitigation.

As used herein, a “visual aid” refers to any solid or liquid materialthat when added to a first mixture provides improved visual contrastbetween the second resultant mixture and the environment to which thesecond mixture is applied. For example, a visual aid may comprise ablack powder, applied to a white first mixture, such that the resultantsecond mixture is gray, so that when the gray mixture is applied to awhite surface, it can be more easily recognized, thus insuring thatappropriate and/or complete coverage of the area to be treated has beenattained. A visual aid may comprise a dye, ink, pigment, bioluminescentbacteria, and combinations thereof. The term “dye” refers to a colorant,usually transparent, which is soluble in an application medium. The term“ink” refers to a liquid or paste containing various pigments and/ordyes used for coloring a surface to produce an image, text, or design.The term “pigment” refers to a synthetic or natural (biological ormineral) material that changes the color of reflected or transmittedlight as the result of wavelength-selective absorption. Further examplesof visual aids are carbon black and fumed silica.

A bioluminescent bioreporter is an organism that is geneticallyengineered to produce light when a particular substance is metabolized.For example, bioluminescent (lux) transcriptional gene fusions may beused to develop light emitting reporter bacterial strains that are ableto sense the presence, bioavailability, and biodegradation of organicchemical pollutants such as naphthalene, toluene, and isopropylbenzene.In general, the lux reporter genes are placed under regulatory controlof inducible degradative operons maintained in native or vector plasmidsor integrated into the chromosome of the host strain. Bioluminescentmicroorganisms are disclosed in U.S. Patent Publication No. 2007/0002994to Simpson et al., which is incorporated by reference herein in itsentirety.

Due to the widespread use of petroleum products and the currentregulations requiring underground storage tanks to be upgraded, replacedor closed, the number of petroleum-contaminated sites has abounded. Ofparticular concern for drinking water quality are the more water-solublecomponents, benzene, toluene, ethylbenzene and xylenes (BTEX). Naturalattenuation which relies on in situ biodegradation of pollutants hasreceived a large amount of attention especially for petroleumcontaminants. While microorganisms capable of biodegradation of BTEXcompounds are usually present at these sites, there is a need to knowwhether or not conditions are favorable for biodegradation to occur.

Bioluminescent reporters have been widely used for the real timenon-destructive monitoring of gene expression. Heitzer et al. (1992)developed a quantitative assay for naphthalene bioavailability andbiodegradation using a nah-lux reporter strain HK44 constructed by Kinget al. (1990) containing a lux transposon (Tn4431) insertion in nahG ofthe lower naphthalene degradation operon. The nah-lux reporter wasexpanded for use as an online optical biosensor for application ingroundwater monitoring (Heitzer et al., 1994). Other lux fusions havebeen constructed for monitoring the expression of catabolic genesincluding those for degradation of isopropylbenzene (Selifonova et al.,1996) and toluene (Applegate et al., 1997).

As used herein, the term “biocatalyst” refers to any enzyme, microbe,protein, amino acid, nucleic acid, fat, lipid or mixture thereof whichis capable of interacting or reacting with contaminants, or is capableof increasing the decomposition and/or degradation rates ofcontaminants.

An aspect of the present invention includes a composition for theremediation of contaminated soils or water supplies comprising an oxidephase, a silicate phase, a pH adjustment agent, a densification agent, anutrient, a surfactant, and a biocatalyst.

In some embodiments of the present invention, the oxide phase maycomprise silica, alumina, iron (II) oxide, iron (II,III) oxide, iron(III) oxide, iron (III) oxide trihydrate, calcium oxide, magnesiumoxide, potassium oxide, and/or combinations thereof. In furtherembodiments, the oxide phase may comprise silica, alumina, iron (III)oxide, and/or calcium oxide. In various embodiments of the invention,the oxide phase may comprise diatomaceous earth (also known Celite,diatomite, and kieselguhr), which typically comprises about 80 to 90%silica, about 2 to 4% alumina, and about 0.5 to 2% iron oxide.Diatomaceous earth is naturally occurring sedimentary rock that iseasily crumbled into a fine white powder, with a typical particle sizein the 10 to 200 μm range. In one embodiment of the present invention,the oxide phase may include Brownmillerite, which typically comprises amixture of calcium oxide, alumina, and iron oxide.

In still further embodiments of the invention, the oxide phase maycomprise fly ash. Fly ash provides the advantage of being readilyavailable from the electrostatic precipitators and bag filters ofcoal-fired power plants because fly ash is a waste product from thermalpower stations. Thus, the present invention provides an outlet and usefor an industrially produced by-product stream. A second advantage isthat fly ash is generally in particulate form consisting of very smallsizes, e.g., 0.5 μm to 300 μm. The small size provides an extremelylarge amount of surface area per unit mass, which facilitates bettercontacting of the fly ash with the other components in the compositionof the present invention, as well as with the target contaminants, whichin turn increases the rates of the decontamination reactions. Pulverizedfly ash, which is used in some embodiments, is typically made of 95%oxides of silicon, aluminum, and iron. The remaining 5% is often unburntcoal and oxides of titanium, potassium, calcium, and other metal oxides.In additional embodiments, cenospheres, which are hollow sphericalparticles with a similar size range as fly ash, can be used with fly ashor in the place of fly ash; however, using cenospheres will increase theprice of the final bioremediation composition. In some embodiments, thefly ash is washed with water and dried before it is added to thebioremediation composition.

In some embodiments of the present invention, the silicate phase maycomprise Belite (Ca₂SiO₄), Alite (Ca₃O·SiO₄), tricalcium silicate(Ca₃SiO₅), and/or combinations thereof. In some embodiments the silicatephase may comprise at least one of cement kiln dust, cement, sol gels,and combinations thereof. In still further embodiments of the presentinvention, the silicate phase may comprise cement kiln dust. In stillfurther embodiments of the present invention, the silicate phase maycomprise Portland cement kiln dust. Cement kiln dust is an advantageousraw material as it is regularly collected in efforts to mitigateemissions from cement kilns. Thus, this source of silicate provides anoutlet for an industrially generated by-product stream. Also, like thefly ash described above, kiln dust comprises a distribution of verysmall particles, with the largest kiln dust particles rarely exceeding0.3 mm. These small particle sizes provide an extremely large amount ofsurface area per unit mass, which facilitates better contacting of thekiln dust with the other components in the composition of the presentinvention, as well as with the targeted contaminants. This in turnincreases the rates of the decontamination reactions. In still furtherembodiments of the present invention, the silicate phase may include anyknown calcium containing silicate mineral, as known by one of ordinaryskill in the art.

In some embodiments of the present invention, the pH adjustment agentmay include at least one of an acid, a base, and a carbonate. In furtherembodiments of the present invention, the pH adjustment agent maycomprise calcium carbonate. Calcium carbonate is a convenient, readilyavailable, and cost effective pH adjustment agent as it is commonlyfound in rock all around the world, including, but not limited tolimestone, chalk, marble, and travertine. An additional advantage isthat these calcium carbonate containing rocks are relatively soft andare readily comminuted and reduced to powder form. Finally, because manynaturally occurring bacteria prefer neutral or basic conditions, calciumcarbonate with a pH value of 9.4 is effective at providing powdercompositions for bioremediation that can elevate the pH of thebioremediation composition as well as the pH of the targetedcontamination site.

In various embodiments of the invention, the composition may furtherinclude one or more of metal, baking soda, bioluminescent materials,perfumes, charcoal, odor absorbing compounds, and clay. In oneembodiment, clay is added to the composition to improve the abrasiveaction of the fly ash and/or to change the color of the composition,e.g., lighten the color of the composition.

In some embodiments of the present invention, the densification agentmay comprise at least one of a powder, a dust, a shaving, a particulate,and combinations thereof, and wherein the densification agent has aparticle density of greater than about 1.5 g/cm³, greater than about 2.0g/cm³, greater than about 2.5 g/cm³, or greater than about 3.0 g/cm³. Insome embodiments of the present invention, the densification agent mayhave a particle density in the range from about 1.5 g/cm³ to about 5.0g/cm³. As used herein, the term “particle density” refers to the densityof the solid excluding the interstitial volume between individualparticles. Including this empty volume contribution results in amaterial's bulk density. Therefore, the bulk density for a powder willbe less than (or equal to if there is no interstitial space) theparticle density. In some further embodiments of the present invention,the densification agent may comprise a metal, a plastic, a mineral, andcombinations thereof. In still further embodiments of the presentinvention, the densification agent may comprise sand. In one embodimentof the invention, the sand used as a densification agent may compriseAndesite, a type of wicking sand, ground quartz, powdered quartz, orwhatever is readily and economically available in a given geographicarea. In some embodiments of the present invention, the sand used as adensification agent, may comprise a particles size in the range fromabout 100 microns to about 1000 microns. The purpose of thedensification agent is to provide the remediation composition with abulk density that is sufficiently high to enable efficient applicationof the composition to the surface, area, volume, etc. that is targetedfor treatment. It will be known to one skilled in the art that theapplication of low density powders such as carbon black and fumed silicais extremely difficult, especially if the application is in an openenvironment subject to variable air flow. Wind losses of thebioremediation product result in the higher application andbioremediation costs. A densification agent alone, or in combinationwith a binding agent, will reduce such product losses and increase theefficiency of the bioremediation process.

In further embodiments of the present invention, the composition mayfurther comprise a binding agent. Without intending to be bound bytheory, a binding agent may provide binding characteristics, wherein twoor more components of the composition are attracted to each other, bydifferences in surface tension, viscosity, charge, polarity, andcombinations thereof. Examples of binding agents include, but are notlimited to, water, polysaccharides, starches, gums, acaia, alginic acid,carboxymethylcellulose, ethylcellulose gelatin, liquid glucose,metylcellulose, povidone, and pregelatinized starch. Examples of gumbinding agents include, but are not limited to, cordial, okra gum,Cassia roxburghii seeds gum, gum Arabic, gum ghatti, ghum tragacanth,and other common gums. In some embodiments of the present invention, thebinding agent may comprise a construction material, for example amortar, a paste, a grout, and combinations thereof. As used herein, theterm “grout” refers to an emulsified mixture comprising water, cement,and sand. A grout may also include fine gravel and a coloring agent.Thus, when a colored grout is used as the binding agent, the grout mayalso serve the purpose of a visual aid.

In some embodiments of the present invention, the composition mayfurther comprise a visual aid selected from the group consisting of adye, ink, pigment, bioluminescent bacteria, and combinations thereof. Infurther embodiments of the present invention, the visual aid may beselected from the group consisting of carbon black, fumed silica andcombinations thereof. In one embodiment, an indicator, such as acolor-change-based indicator can be included in the bioremediationcomposition. The color-change-based indicator may make thebioremediation composition one color initially and then change colorsonce the area to be cleaned is clean. Additionally, developers couldalso be added to the bioremediation composition depending on the type ofindicator used. For example, if the indicator is phenolphthalein, thedeveloper can be an acid or a base depending upon whether the indicatoris used in an acidic or basic substrate. Indicators and developerssimilar to those disclosed in U.S. Pat. No. 6,814,816 to Archar et al.,which is incorporated by reference herein in its entirety, can be usedin some embodiments.

In some embodiments of the present invention, the surfactant maycomprise at least one of a nonionic surfactant, a cationic surfactant,an anionic surfactant, an amphoteric surfactant, and combinationsthereof.

In some embodiments of the present invention, the biocatalyst maycomprise at least one of an enzyme, a bacterium, a fungus, a yeast, andcombinations thereof. In some embodiments of the present invention, thebiocatalyst may comprise at least one bacterium from the Bacillus genus,the Micrococcus genus, and combinations thereof.

An aspect of the present invention is a composition for remediation ofcontaminated liquids or solids comprising an oxide phase between about20 wt % and about 40 wt %, a silicate phase between about 5 wt % andabout 25 wt %, a pH adjustment agent between about 1 wt % and about 20wt %, and a densification agent between about 1 wt % and about 20 wt %.The composition further comprises a nutrient between about 1 wt % andabout 10 wt %, a surfactant between about 1 wt % and about 10 wt % and abiocatalyst between about 10 wt % and about 30 wt %.

In some embodiments of the present invention, the composition mayfurther comprise a visual aid between about 0.1 wt % and about 10 wt %.In some embodiments of the present invention, the composition mayfurther comprise a binding agent between about 0.1 wt % and about 10 wt%.

In some embodiments of the present invention, the composition maycomprise a powder with a particle size of less than 0.125 inches. Insome embodiments of the present invention, the composition may comprisea powder with a particle size of less than 0.0625 inches. In somefurther embodiments of the present invention, the composition maycomprise a powder with a particle size of less than 1000 microns. Insome further embodiments of the present invention, the composition maycomprise a powder with a particle size of less than 100 microns.

In some embodiments of the present invention, the composition maycomprise a moisture content of less than 20 wt % water. In someembodiments of the present invention, the composition may comprise amoisture content of less than 15 wt % water. In some further embodimentsof the present invention, the composition may comprise a moisturecontent of less than 10 wt % water. In some further embodiments of thepresent invention, the composition may comprise a moisture content ofless than 5 wt % water.

An aspect of the present invention is a composition for remediation ofcontaminated liquids or solids comprising between about 20 wt % andabout 40 wt % fly ash, between about 10 wt % and about 20 wt % Portlandcement kiln dust, between about 5 wt % and about 15 wt % sand, betweenabout 0.5 wt % and about 4 wt % milled whole kernel corn, between about0.5 wt % and about 4 wt % milled cotton hulls, between about 5 wt % andabout 15 wt % calcium carbonate, between about 2 wt % and about 10 wt %grout, between about 1 wt % and about 10 wt % detergent, and betweenabout 10 wt % and about 30 wt % of biocatalyst. The biocatalystcomprises a composition of between about 5 wt % and about 15 wt %Bacillus amyloliquifaciens, between about 5 wt % and about 15 wt %Bacillus atophaeus, between about 5 wt % and about 15 wt % Bacillusbenzeovorans, between about 5 wt % and about 15 wt % Bacillus cereus,between about 5 wt % and about 15 wt % Bacillus lichenformis, betweenabout 5 wt % and about 15 wt % Bacillus megarterium, between about 20 wt% and about 25 wt % Bacillus subtilus, between about 5 wt % and about 15wt % Bacillus polymyxa, between about 5 wt % and about 15 wt %Micrococcus flavus, and between about 5 wt % and about 15 wt %Micrococcus conglomerates. The solid remediation composition may furthercomprise a particle size of less than 0.125 inches.

One embodiment of the present invention includes a composition forremediation of contaminated liquids or solids which may comprise about31 wt % fly ash, about 16 wt % Portland cement kiln dust, about 10 wt %sand, about 2 wt % milled whole kernel corn, about 2 wt % milled cottonhulls, about 10 wt % calcium carbonate, about 4 wt % colored grout,about 5 wt % detergent, and about 20 wt % of biocatalyst. Thebiocatalyst may comprise about 9.1 wt % Bacillus amyloliquifaciens,about 9.1 wt % Bacillus atophaeus, about 9.1 wt % Bacillus benzeovorans,about 9.1 wt % Bacillus cereus, about 9.1 wt % Bacillus lichenformis,about 9.1 wt % Bacillus megarterium, about 18.5 wt % Bacillus subtilus,about 9.1 wt % Bacillus polymyxa, about 9.1 wt % Micrococcus flavus, andabout 9.1 wt % Micrococcus conglomerates. One skilled in the art willrecognize that obtaining precise percentage contributions in thecomposition, for any of the microbes listed above, is difficult and thatsome natural variability will occur around the preferred target values.Therefore, compositions with microbe concentrations that fall within areasonable variability range, for example due to the method of producingthe biocatalyst, are intended to fall within the scope of the presentinvention. One skilled in the art will recognize that a microbecomposition may also include residue (e.g., growth media) from themicrobe manufacturing steps. In some embodiments of the presentinvention, the biocatalyst compositions describe above may contain fromabout zero weight percent up to about 75 wt % residual growth media.

Some embodiments of the invention include a composition for thebioremediation of contaminated solids or liquids comprising about 31 wt% fly ash; about 16 wt % kiln dust; about 10 wt % sand; about 10 wt %calcium carbonate; about 5 wt % detergent; about 4 wt % grout; about 2wt % corn; about 2 wt % cotton; and about 20 wt % of a biocatalystmixture comprising: about 9 wt % Bacillus amyloliquifaciens; about 9 wt% Bacillus atophaeus; about 9 wt % Bacillus benzeovorans; about 9 wt %Bacillus cereus; about 9 wt % Bacillus lichenformis; about 9 wt %Bacillus megarterium; about 19 wt % Bacillus subtilus; about 9 wt %Bacillus polymyxa; about 9 wt % Micrococcus flavus; and about 9 wt %Micrococcus conglomeratus.

In some embodiments of the present invention, the bioremediationcomposition consists of a two-part formulation, wherein a first partconsisting of all of the remaining non-biocatalyst components, e.g.,oxide phase, a silicate phase, a pH adjustment agent, a densificationagent, a nutrient, a surfactant, binding agent, and visual aid, and asecond part consists of the biocatalyst with or without residual growthmedia. In some embodiments of the present invention, a mass ratio of thesecond part to the first part may be in the range from about 0.1 to 1.0,to about 10 to 1.0.

In some embodiments of the present invention, the solid bioremediationcomposition may be converted to a liquid formulation by mixing withwater. In some embodiments of the present invention, about one pound ofthe solid bioremediation composition is mixed with about one gallon ofwater, resulting in a final liquid form bioremediation product. In someembodiments of the present invention, a ratio of the composition toliquid water is in the range of about 0.1 pounds of solid to about onegallon of water, to about 10 pounds of solid to about one gallon ofwater.

The following paragraphs describe methods to manufacture thecompositions of the present invention, in order to enable one ofordinary skill in the art to make and use such compositions. For powderand/or granular compositions, various methods known to one of ordinaryskill in the art may be used. In some embodiments of the presentinvention, the desired composition can be achieved by individuallyweighing out each component followed by transfer to an industrial mixeror blender, wherein they are subsequently blended. So, any of thecomponents of the remediation composition, e.g., oxide phase, silicatephase, pH adjustment agent, densification agent, nutrient, surfactant,and biocatalyst, may be mixed together using an industrial mixer orblender. In some embodiments of the present invention, the mixer orblender may be positioned on a weigh-scale and/or weigh-cells, so thateach component can be added directly to the mixing device, thuseliminating a separate weigh step and transfer step, and thus providinga more reliable and accurate batching process. Examples of blenders andmixers that may be used to formulate the compositions of the presentinvention include, but are not limited to, ribbon blenders, V-blenders,cone screw blenders, screw blenders, double cone blenders, planetarymixers, dispersion mixers, counter-rotating mixers, paddle mixers, jetmixers, drum blenders, Banbury mixers, and combinations thereof.

Mixing may be achieved in a batch mixing process or in a continuousprocess. A single mixing device may be used, or two or more in seriesand/or in parallel may be used. The various solid components may beadded using standard means known to one of ordinary skill in the art,for example single-screw or twin-screw extruders. In addition, themixers may or may not provide temperature and pressure controls.

In some embodiments of the present invention, a liquid processing aidmay be used to facilitate more complete and/or faster mixing of thebioremediation composition components. Examples of liquid additives thatmay be used include water, alcohols and solvents. In some embodiments ofthe present invention, less than all of the composition components maybe mixed in a liquid processing aid, creating an intermediate dispersedphase. After mixing is achieved the liquid processing aid may beevaporated to create a first part of the composition. This may then besubsequently added to a second part of the composition, either duringsubsequent manufacturing steps, or during the application step to thecontaminated area. For example, an oxide phase, a silicate phase, a pHadjustment agent and a densification agent may be mixed together inmethanol to create a well mixed dispersion, which is subsequently driedusing heat and/or vacuum, to create a first solid part of thecomposition. A second solid part is separately prepared, in a separatemixer, comprising a nutrient, a surfactant, and a biocatalyst. The twoparts may then be added together in a subsequent manufacturing step, orat the contamination site. The concept of a two-part formulation alsoapplies to a system where neither part undergoes a liquid mixing stepand/or evaporation step, or where both parts undergo a liquid mixingstep and evaporation step. In addition, the composition may comprisemore than a two-part formulation, e.g., a three-part formulation ormore.

Liquid formulations may be achieved in standard equipment known to oneof ordinary skill in the art, in either batch or continuous processes.Such equipment includes for example, stirred-tank reactors, continuousstirred-tank reactors, and static mixers. Mixers for liquid formulationmay also include temperature and pressure control systems.

The biocatalyst component may also be manufactured using standardmethods and equipment known to one of ordinary skill in the art. In oneembodiment of the present invention, for biocatalysts comprising morethan one bacterial strain, each strain may be individually grown andsubsequently lyophilized. The final desired biocatalyst may then beachieved by mixing the individually lyophilized strains together, at thedesired ratios, much like described above for the non-biocatalystcomponents of the remediation composition. Alternatively, two or more ofthe bacterial strains may be grown together, provided the optimum growthconditions and media are indentified that allow multiple stains toco-exist without outcompeting one another. This approach offers thepotential advantage of requiring less fermentation equipment, fasterproduction rates, and lower manufacturing costs. The same principlesjust highlighted above for growing bacteria, also apply to yeast andfungi.

Various methods of creating the bioremediation composition can be used.In one embodiment, blending of the composition is performed in a paddlemixer at 500 lbs to 1000 lbs mixes. The method comprises creating amicrobial mixture; adding fly ash, kiln dust, sand, calcium,sand-colored grout, and detergent to the paddle mixer; mixing thesecomponents for at least five minutes to create a first compositionmixture; adding cotton, corn, and the microbial mixture to the paddlemixer with the first composition mixture; mixing these components atleast five minutes to create the final bioremediation composition;discharging the final bioremediation composition in less than tenminutes; placing the final bioremediation composition in 1-gallon or5-gallon containers; placing a desiccant bag in the containers with thefinal bioremediation composition; and sealing the containers to keep thefinal bioremediation composition from activating.

The following section provides detail regarding methods for applying theremediation composition of the present invention to a contaminated site.Some embodiments of the present invention may comprise in situ and exsitu bioremediation methods of contaminated solids and soils, wherein insitu techniques are defined as those that are applied to soil andgroundwater at the site with minimal disturbance. Ex situ techniques arethose that are applied to soil and groundwater at the site which hasbeen removed from the site via excavation (soil) or pumping (water).

In one embodiment, a method of ameliorating a contaminated area isprovided, comprising: providing a bioremediation composition comprising:between about 20 wt % to about 40 wt % fly ash; between about 10 wt % toabout 20 wt % kiln dust; between about 5 wt % and about 15 wt % sand;between about 0.5 wt % and about 4 wt % corn; between about 0.5 wt % andabout 4 wt % cotton; between about 5 wt % and about 15 wt % calciumcarbonate; between about 2 wt % and about 10 wt % grout; between about 1wt % and about 10 wt % detergent; and between about 10 wt % and about 30wt % of a biocatalyst mixture; spreading an effective amount of thebioremediation composition onto the contaminated area, wherein thecontaminated area comprises a first color characteristic; and providingan effective amount of time in which the bioremediation composition caninteract with the contaminated area, wherein after the effective amountof time the contaminated area comprises a second color characteristic,and wherein the second color characteristic is different than the firstcolor characteristic. In a further embodiment, the bioremediationcomposition further comprises a bioluminescent microbe, and the secondcolor characteristic results from a decrease in bioluminescenceassociated with the bioremediation composition. In one embodiment, aneffective amount of time is at least one day. In a preferred embodiment,an effective amount of time is between two days and 30 days. In a morepreferred embodiment, an effective amount of time is between five daysand 14 days. In another embodiment, an effective amount of time is fivedays.

In situ techniques are generally the most desirable options due to lowercost and fewer disturbances to the environment since they provide thetreatment in place and avoid excavation and transport of contaminatedmass. In situ treatment may be limited by the depth of the soil that canbe effectively treated. In many soils effective oxygen diffusion fordesirable rates of bioremediation extend to a range of only a fewcentimeters to about 30 cm into the soil, although depths of 60 cm andgreater have been effectively treated in some cases. In some embodimentsof the present invention, an in situ technique involves mechanicallyspreading a solid remediation composition of the present invention ontothe contaminated surface. This may be performed using a standardspreader device known to one of ordinary skill in the art. In someembodiments, a single spreading step may complete the applicationprocess, wherein all of the components are included in a singleformulation. In other embodiments, which use two- or multiple-partformulations, multiple spreading steps may be used. In one embodiment,the bioremediation composition may be rubbed, brushed, or worked intothe surface or ground to be cleaned using a mechanical action to workthe bioremediation composition into the pours or grains of the surfaceand/or to spread the bioremediation composition around the contaminatedarea. In still further embodiments, when applied to solid surfaces, theapplication of a remediation composition may be subsequently followed bywetting the composition with water. The water may be applied as a spray,using standard methods known to one of ordinary skill in the art. Otherliquid wetting agents and wetting formulations may also be used.

Further examples of in situ techniques that may be utilized in someembodiments of the present invention include bioventing, biodegradation,biosparging, and bioaugmentation. Bioventing involves supplying air andnutrients through wells to contaminated soil. In some embodiments of thepresent invention, pressurized air may be used as a pneumatic carriergas to transport a solid and/or liquid remediation composition of thepresent invention to subsurface contamination zones, such as watersupplies and aquifers.

In situ biodegradation typically involves supplying oxygen and nutrientsby circulating aqueous solutions through contaminated soils to stimulatenaturally occurring bacteria to degrade contaminants. It can be used forsoil and groundwater. Some embodiments of the present invention mayinclude conditions such as the infiltration of water-containingnutrients and oxygen or other electron acceptors for groundwatertreatment, after application of the solid or liquid bioremediationcomposition of the present invention. The bioremediation composition maybe initially applied, for example, by tilling.

In situ biosparging typically involves the injection of air underpressure below the water table to increase groundwater oxygenconcentrations and enhance the rate of biological degradation ofcontaminants by naturally occurring bacteria. Biosparging increases themixing in the saturated zone and thereby increases the contact betweensoil and groundwater. The ease and low cost of installing small-diameterair injection points allows considerable flexibility in the design andconstruction of the system. In some embodiments of the presentinvention, the pressurized air of a biosparging process may act as acarrier gas to pneumatically convey a powdered and/or liquid remediationcomposition of the present invention to a subsurface water source, forexample an aquifer.

Ex situ techniques typically involve the excavation or removal ofcontaminated soil from the ground. Examples of ex situ bioremediationtechniques that may be used in some embodiments of the present inventioninclude land-farming, composting, biopiles, and bioreactors.

Ex situ landfarming is a technique in which contaminated soil isexcavated and spread over a prepared bed and periodically tilled untilpollutants are degraded. The goal is to stimulate indigenousbiodegradative microorganisms and facilitate their aerobic degradationof contaminants. In general, the practice is limited to the treatment ofsuperficial 10-35 cm of soil. Since landfarming has the potential toreduce monitoring and maintenance costs, as well as clean-upliabilities, it has received much attention as a disposal alternative.In some embodiments of the present invention, the remediationcompositions of the present invention may be applied to the preparedbeds, in at least one application, followed by periodic tillage. Thecomposition may supplement the indigenous microorganisms, potentiallyresulting in faster and more complete remediation of the pollutants.

Ex situ composting is a technique that involves combining contaminatedsoil with nonhazardous organic amendants such as manure or agriculturalwastes. The presence of these organic materials supports the developmentof a rich microbial population and elevated temperature characteristicof composting. Similar to the landfarming example given above, in someembodiments of the present invention, compositions of the presentinvention may be combined with composting methods to create moreeffective and faster bioremediation of contaminated sites.

Ex situ biopiles are a hybrid of landfarming and composting.Essentially, engineered cells are constructed as aerated compostedpiles. Typically used for treatment of surface contamination withpetroleum hydrocarbons they are a refined version of landfarming thattend to control physical losses of the contaminants by leaching andvolatilization. Biopiles provide a favorable environment for indigenousaerobic and anaerobic microorganisms. The present invention is ideallysuited to supplement and improve the bioremediation of contaminantsusing biopiles.

Examples of bioreactors include slurry reactors or aqueous reactors areused for ex situ treatment of contaminated soil and water pumped up froma contaminated plume. Bioremediation in reactors involves the processingof contaminated solid material (soil, sediment, sludge) or water throughan engineered containment system. A slurry bioreactor may be defined asa containment vessel and apparatus used to create a three-phase (solid,liquid, and gas) mixing condition to increase the bioremediation rate ofsoil-bound and water-soluble pollutants as a water slurry of thecontaminated soil and biomass (usually indigenous microorganisms)capable of degrading target contaminants. In general, the rate andextent of biodegradation are greater in a bioreactor system than in situor in solid-phase systems because the contained environment is moremanageable and hence more controllable and predictable. In someembodiments of the present invention, the presently disclosedcompositions are used to increase the efficiency and reaction rates ofcontaminant decomposition reactions in bioreactors.

In some embodiments of the invention, a method of producing abioremediation composition using biological hosts tosynthesize/accumulate bioproducts such as bacteria, eukaryoticmicroorganisms (e.g., Saccharomyces cerevisiae), plants, animal cells(e.g., transformed insect cells growing in culture), and animals isprovided. Thus, methods by which effector-sensitive RCANAs can be usedto facilitate industrial biosynthesis and bioremediation. For example,provided herein are methods in which effector-dependent ribozymes can beused to (1) control production of a natural product in a biologicalhost, (2) to identify environmental conditions which increasebiosynthetic yields, and (3) to isolate strains of a biological hostwith improved product yields and/or properties. RCANA are more robustthan allosteric protein enzymes in several ways: (1) they can beselected in vitro, which facilitates the engineering of particularconstructs; (2) the levels of catalytic modulation are much greater forRCANA than for protein enzymes; and (3) because RCANA are nucleic acids,they can potentially interact with the genetic machinery in ways thatprotein molecules may not.

Various embodiments of the present invention include RCANAs where thecatalytic activity of the RCANA is regulated by an effector. The RCANAare, therefore, regulatable in that their activity is under the controlof a second portion of the RCANA. Just as allosteric protein enzymesundergo a change in their kinetic parameters or of their enzymaticactivity in response to interactions with an effector, the catalyticabilities of the RCANA may similarly be modulated by the effector(s).Thus, some embodiments of the invention are directed to RCANA thattransduce molecular recognition into catalysis. Also, RCANAs can be usedas regulatory elements to control the expression of one or more genes ina metabolic pathway. RCANAs can also be used as regulated selectablemarkers to create a selective pressure favoring (or disfavoring)production of a targeted bioproduct.

The methods may include any type of nucleic acid. For example, themethods are not limited to RNA-based RCANA, but also encompass DNA RCANAand RNA or DNA RCANA. Furthermore, the methods can be applied to anycatalytic activity the ribozymes are capable of carrying out. Forexample, the methods are not limited to ligases or splicing reactions,but could also encompass other ribozyme classes. The methods are alsonot limited to protein or peptide ligands, but also include-othermolecular species, such as ions, small molecules, organic molecules,metabolites, sugars and carbohydrates, lipids and nucleic acids. Themethods may also be extended to effectors that are not molecules, suchas heat or light or electromagnetic fields. Furthermore, the methods arenot limited to ligand-induced conformational changes, but could alsotake into account chimeric catalysts in which residues essential forchemical reactivity, were provided by both the nucleic acid and theligand, in concert. Additionally; the effector may be a peptide, apolypeptide, a polypeptide complex, or a modified polypeptide orpeptide. The effector may even be, e.g., an enzyme or even light (suchas visible light) or even a magnet. The effector may be activated by asecond effector that acts on the first effector, which may be aninorganic or an organic molecule. The polypeptide, peptide orpolypeptide complex can be either endogenous, i.e., derived from thesame cell type as the polynucleotide, or exogenous, i.e., derived from acell type different than the cell from which the polynucleotide isderived.

In another embodiment of the present invention, a method of detecting anion in the presence of other ions in a sample is provided. The methodcomprises: forming a mixture of a nucleic acid enzyme including at leastone quencher, a substrate and the sample, to produce a product; anddetecting the presence of the product. The substrate is a nucleic acidsequence including a ribonucleotide, at least one quencher and at leastone fluorophore. A “nucleic acid enzyme” is a nucleic acid molecule thatcatalyzes a chemical reaction. The nucleic acid enzyme may be covalentlylinked with one or more other molecules yet remain a nucleic acidenzyme. Examples of other molecules include dyes, quenchers, proteins,and solid supports. The nucleic acid enzyme may be entirely made up ofribonucleotides, deoxyribonucleotides, or a combination of ribo- anddeoxyribonucleotides.

In another embodiment of the invention; a method of determining theconcentration of an ion in the presence of other ions, in a sample,comprising: forming a mixture of a nucleic acid enzyme comprising atleast one quencher, a substrate comprising a ribonucleotide, at leastone quencher and at least one fluorophore, and the sample, to produce aproduct; and measuring the amount of product produced.

In various embodiments of the invention, a biosensor, capable ofdetecting the presence of an ion in the presence of other ions, isprovided comprising: a nucleic acid enzyme which includes at least onequencher, and a substrate which includes a ribonucleotide, at least onequencher and at least one fluorophore. A growing number of nucleic acidenzymes have been discovered or developed showing a great diversity incatalytic activity. Many, if not all, of the enzymes are dependent onone or more ion cofactors. In vitro selection may be used to “enhance”selectivity and sensitivity for a particular ion. Such enzymes findparticular utility in the compositions and methods of the presentinvention. For example, nucleic acid enzymes that catalyze molecularassociation (ligation, phosphorylation, and amide bond formation) ordissociation (cleavage or transfer) are particularly useful. In someembodiments, a nucleic acid enzyme that catalyzes the cleavage of anucleic acid in the presence of an ion is used. The nucleic acid enzymemay be RNA (ribozyme), DNA (deoxyribozyme), a DNA/RNA hybrid enzyme, ora peptide nucleic acid (PNA) enzyme. PNAs comprise a polyamide backboneand the bases found in naturally occurring nucleosides and arecommercially available, e.g., from Biosearch, Inc. in Bedford, Mass.Similar biosensors are described in U.S. Pat. No. 7,906,320 to Lu etal., which is incorporated by reference herein in its entirety.

In certain embodiments, the present invention is directed to abio-remediate formulation containing Sodium/CalciumEthylenediaminetetraacetic acid (EDTA), for the cleanup of soil,concrete, asphalt, metal, wood, ceramic, stone, other hard surfaces andincluding water, that have been contaminated with petroleum hydrocarbonsand or mine tailings containing toxic metals. EDTA holds and collectsthe metals and allows the bio-remediation formula to attack andbreakdown the metals that are in petroleum hydrocarbons and minetailings.

One embodiment is directed to a soil and hard surfaces formulation. Byadding 5 to 20% of EDTA to a 500 lbs mix of the present bio-remediationformula. EDTA holds the metal letting the bio-remediate formula attackthe metals. Contamination is cleaned up in 30 to 180 days. Using lowerpercentages of EDTA from 5 to 9% in the bioremediation formula, thebio-remediation time was 180 days. Increasing the EDTA levels 10 TO 14%Butin the same formula, bio-remediation time was decreased to 90 days.Increasing the EDTA levels 15 to 20%, the bio-remediation time wasdecreased to 30 days.

No difference in increasing the levels of EDTA higher than 20% to the500 lbs. mix for this application was seen as beneficial.

Tape pull testing may be performed to evaluate the success of the metalsbio-remediated.

Another embodiment is directed to a water formulation. The cottoncontent is preferably increased (ground cotton hulls 90 lbs., andcottonseed meal 50 lbs. and 100 lbs. commercial finally of ground sand,add in 50 lbs. of the bio-remediation formula plus 20% of Sodium/CalciumEthylenediaminetetraacetic acid) (EDTA), and placing of the formulationin a chemical spill absorbent sock material and or mesh material (in 4inch, 6 inch, 8 inch, and 12 inch diameter sizes in length), the socksmay be used around storm drains.

In water placing chemical resistant flotation devices in the center ofthe chemical spill absorbent sock material (ranging from 2 inch to 8inch in size), filled with the EDTA Bio-remediation Formula, givespositive results. The spill sock can also be fastened to flotation buoysas needed to achieve proper water depth. The spill sock allows the waterto flow through while collecting the oil/metal or metal contaminationand letting the bio-remediation process to begin.

In other embodiments, metals in vehicle fluids are contained in theabove-mentioned socks. As motor fluids circulates through vehicleengines, steering, braking, hydraulic and cooling systems picking upheavy metals such as arsenic, lead, cadmium, copper and zinc, as well asother toxins. Main tailings, such as the contamination that entered theAnimas River in Colorado in Sep. 8, 2015, can be contained using thepresent invention.

Various metals, including Arsenic (As), Barium (Ba), Cadmium (Cd),Chromium (Cr), Lead (Pb), Mercury (Hg), Selenium (Se), Silver (Ag) andThallium (Tl) can also be contained, as well as all the below listedelements/components:

EPA (The D List)

EPA Hazardous Regulated Waste Level (mg/l) code Contaminant (or ppm)D004 Arsenic (As) 5.0 D005 Barium (Ba) 100.0 D018 Benzene 0.5 D006Cadmium (Cd) 1.0 D019 Carbon Tetrachloride 0.5 D020 Chlordane 0.03 D021Chlorobenzene 100.0 D022 Chloroform 6.0 D007 Chromium (Cr) 5.0 D023o-Cresol 200.0 D024 m-Cresol 200.0 D025 p-Cresol 200.0 D026 Cresol 200.0D016 2,4-D 10.0 D027 1,4-Dichlorobenzene 7.5 D028 1,2-Dichloroethane 0.5D029 1,1-Dichloroethylene 0.7 D030 2,4-Dinitrotoluene 0.13 D012 Endrin0.02 D031 Heptachlor 0.008 D032 Hexachlorobenzene 0.13 D033Hexachlorobutadiene 0.5 D034 Hexachloroethane 3.0 D008 Lead (Pb) 5.0D013 Lindane 0.4 D009 Mercury (Hg) 0.2 D014 Methoxychlor 10.0 D035Methyl ethyl ketone 200.0 D036 Nitrobenzene 2.0 D037 Pentachlorophenol100.0 D038 Pyridine 5.0 D010 Selenium (Se) 1.0 D011 Silver (Ag) 5.0 D039Tetrachloroethylene 0.7 D015 Toxaphene 0.5 D040 Trichloroethylene 0.5D041 2,4,5-Trichlorophenol 400.0 D042 2,4,6-Trichlorophenol 2.0 D0172,4,5-TP (Silvex) 1.0 D043 Vinyl Chloride 0.2

All publications, patents, and patent documents cited herein areincorporated by reference herein, as though individually incorporated byreference. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention that are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany sub-combination.

The invention now being generally described will be more readilyunderstood by reference to the following examples, which are includedmerely for the purposes of illustration of certain aspects of theembodiments of the present invention. The examples are not intended tolimit the invention, as one of skill in the art would recognize from theabove teachings and the following examples that other techniques andmethods can satisfy the claims and can be employed without departingfrom the scope of the claimed invention.

EXAMPLES

Laboratories produce so-called “ideal” conditions which may be “ideal”for very short times in small-scale situations. However, full-scale,real-life, field work, involves highly variable environments that may beactually more ideal than small-scale systems. The laboratory conditionsused to evaluate microbial activities are often irrelevant in thecontext of real-life remediation conditions. Most commercial microbialapplications deal with field conditions not typically found in thelaboratory. Many everyday products we currently use are essentiallybetter designed modifications of existing technology. For a product togain acceptance, it must have new benefits when used in a wide varietyof existing conditions. How the product may have performed under rigidlycontrolled, unchanging laboratory conditions is often a poor predictionof how the product may perform in real-life. However, the followingexamples are provided to provide further disclosure and enablement:

Example 1: One embodiment of the present invention includes abioremediation composition with a first part and a second part. Thefirst part may be prepared by mixing the following components:

TABLE 1 Component Mass [lb] % Fly ash 235 44.8 Kiln dust 120 22.9Calcium carbonate 50 9.5 Sand 50 9.5 Milled corn kernels 10 1.9 Milledcotton hulls 10 1.9 Colored grout 15 2.9 Detergent 35 6.7 Total 525 100%

The corn and cotton hulls may be milled by any suitable method, forexample, using a hammer mill. The mixer may be placed on weigh cells,with each component added directly to the mixer, in order to achieve thetarget mass for each component.

The second part of the bioremediation composition may be manufacturedseparately from the first part, where the second part comprises abiocatalyst. The biocatalyst can comprise the following components:

TABLE 2 Component Mass [lb] % Bacillus amyloliquifaciens 9.1 9.1%Bacillus atophaeus 9.1 9.1% Bacillus benzeovorans 9.1 9.1% Bacilluscereus 9.1 9.1% Bacillus lichenformis 9.1 9.1% Bacillus megarterium 9.19.1% Bacillus subtilus 18.5 18.5%  Bacillus polymyxa 9.1 9.1%Micrococcus flavus 9.1 9.1% Micrococcus conglomeratus 9.1 9.1% Total 100100% 

The second part of the composition may be produced by individuallygrowing each species of bacteria in its own separate culture, withgrowth media optimized for its particular metabolic needs. This may bedone using standard methods known to one of ordinary skill in the art ofbacterial fermentations, for example, stage-wise culturing toprogressively larger fermentation vessels, to produce quantities largeenough for large-scale manufacturing processes. After each fermentationis completed, the organisms may be lyophilized to produce large drymaster-batches of each individual species. Alternatively, the organismscan be dried using other drying methods now known or later developed.These individual master-batches may then be mixed in a dry mixer, muchlike the first part, to the desired formulation as specified in Table 2.The completed microbe composition may then be packaged for eventualcombination with the first part of the total composition. This may bedone in the same mixer used to produce the first part, or in a differentmixer. Once the first and second parts have been sufficiently mixed, thecomplete bioremediation composition may be packaged as needed.

Example 2: Some embodiments of the present invention include abioremediation composition with the following components:

TABLE 3 % in % in % in Range Range Final Raw Final High Low Raw MaterialProduct Substance Material Product (Est.) (Est.) Fly ash 31 Silicondioxide 60 18.6 19.5 17.7 Fly ash 31 Aluminum oxide 20 6.2 6.5 5.9 Flyash 31 Calcium oxide 10 3.1 3.2 2.9 Fly ash 31 Iron III 7 2.17 2.3 2.1Microbial broth 20 100 20 21 19 Kiln dust 16 Calcium oxide 65 10.4 10.99.9 Kiln dust 16 Silicon dioxide 15 2.4 2.5 2.3 Kiln dust 16 Sulfurtrioxide 5 0.8 0.8 0.76 Kiln dust 16 Aluminum oxide 4 0.64 0.6 0.57 Kilndust 16 Potassium oxides 4 0.64 0.6 0.57 Calcium carbonate 10 Calciumcarbonate 100 10 10.5 9.5 Sand 10 Silicon dioxide 96 9.6 10.1 9.1 Grout4 Silicon dioxide 55 2.2 2.3 2.1 Grout 4 Calcium oxide 20 0.8 0.8 0.76Grout 4 Aluminum oxide 18 0.7 0.74 0.66 Detergent 5 Sodium carbonate 291.45 1.5 1.3 Detergent 5 Sodium chloride 29 1.45 1.5 1.3 Detergent 5Acrylic Polymer 15 0.75 0.8 0.76 Detergent 5 Cellulose Gum 15 0.75 0.80.76 Corn 2 Corn 100 2 2.1 1.9 Cotton 2 Cotton 100 2 2.1 1.9

The raw product in the first column is comprised of the substanceslisted in the third column from the left. Thus, the percentage of thesubstance in the final product is listed in the fifth column.Additionally, a range of the substances' percentages is given in thelast two columns on the right.

What is claimed is:
 1. A method of treating contaminated soil or watersupply, comprising: providing a bioremediation composition comprising:between about 20 wt % and about 40 wt % fly ash; between about 10 wt %and about 20 wt % kiln dust; between about 5 wt % and about 15 wt %sand; between about 0.5 wt % and about 4 wt % corn; between about 0.5 wt% and about 4 wt % cotton; between about 5 wt % and about 15 wt %calcium carbonate; between about 2 wt % and about 10 wt % grout; betweenabout 1 wt % and about 10 wt % detergent; between about 10 wt % andabout 30 wt % of a biocatalyst mixture; and at least one bioluminescentbioreporter comprising a microbe comprising Saccharomyces cerevisiaeemploying yeast expression vector pYES2; spreading an effective amountof the bioremediation composition onto the contaminated soil or watersupply, wherein the contaminated soil or water supply comprises a firstcolor characteristic; and providing an effective amount of time in whichthe bioremediation composition can interact with the contaminated soilor water supply, wherein after the effective amount of time thecontaminated soil or water supply comprises a second colorcharacteristic, wherein at least a portion of the bioremediationcomposition is contained in material that allows for water to flowthrough.